We are using the proteins encoded by the E. Coli bacteriophage T4 as a model to study the types of protein-protein interactions and enzymatic reactions required for duplex DNA replication. Efficient DNA replication in vitro is achieved with T4 DNA polymerase (gene 43), gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, the genes 41, 61, and 59 primase- helicase, RNase H, and DNA ligase. We are collaborating with Timothy Mueser and Craig Hyde, NIAMS, to determine the structure of the T4 DNA replication proteins by X-ray diffraction. The bacteriophage T4 gene 59 helicase assembly protein is required for recombination-dependent DNA replication, which is the predominant mode of DNA replication in the late stage of T4 infection. 59 Protein accelerates the loading of the T4 gene 41 helicase during DNA synthesis by the T4 replication system in vitro. It has been shown to bind to both T4 gene 41 helicase and gene 32 single-stranded DNA binding protein, and to single- and double- stranded DNA. We find that T4 gene 59 protein binds most tightly to fork DNA substrates, with either single- or almost entirely double- stranded arms. Our studies suggest that the helicase assembly protein is responsible for loading T4 gene 41 helicase specifically at replication forks, and that its binding sites for each arm must hold more than 6, but not more than 12 nucleotides. In addition, we find that 59 protein facilitates the binding of 32 protein near the fork. Our collaborators, Timothy Mueser and Craig Hyde have determined a high resolution crystal structure of the full length 217 residue monomeric 59 protein, which has a novel alpha helical bundle fold with two domains of similar size. 59 Protein represents a new class of DNA binding protein, since its overall fold does not match any previous structure. We have proposed a speculative model of how this helicase assembly protein might bind to fork DNA, the helicase and 32 protein, based on the structural similarity of its N-terminal domain to the double-stranded DNA binding motif of rat HMG1, the location of its surface basic, acidic, and extensive hydrophobic regions, and the site size of the fork arms needed for tight fork DNA binding. We have constructed site-directed mutations in 59 protein that we are using to test this model. T4 RNaseH is a 5 to 3 exonuclease that is a member of the RAD2 family of eukaryotic and prokaryotic replication and repair nucleases. We have shown that this nuclease removes the pentanucleotide RNA primers and 10-50 nucleotides of adjacent DNA from each discontinuous lagging strand fragment on the DNA replication fork in vitro. The extent of DNA removal by the nuclease is regulated by gene 32 protein. On nicked and gapped model substrates for DNA repair, DNA degradation is stimulated by loading the gene 45 polymerase clamp protein behind the nuclease. We are using wild type and mutant T4 RNaseH to study its interactions with 32 protein and the polymerase clamp. Two bacteriophage T4 origins of replication, (ori(uvsY) and ori(34)), have been shown by Ken Kreuzer and coworkers to consist of a middle-mode promoter juxtaposed to a DNA unwinding element (DUE). In vivo, the origin transcript at ori(uvsY) forms a stable R-loop within the DUE region. In collaboration with Kreuzer and Kathleen Dudas (Duke University) we have established an in vitro system for the extension of preformed R-loops on supercoiled plasmids with the uvsY origin. In the presence of T4 DNA topoisomerase as well as the polymerase, clamp, and clamp loader; the primase, helicase, and helicase assembly protein; and 32 protein, replication proceeds in a unidirectional manner resulting in the rapid formation of full-length (5.7kb) products. The lagging strand fragments of 1-2 kb are sealed upon addition of T4 RNase H and DNA ligase, forming predominantly supercoiled monomeric products. Replication of the synthetic R-loop template in the absence of T4 topoisomerase arrests with a truncated leading strand of about 1.5 kb. We have begun a collaboration with the laboratory of Jack Griffith (University of North Carolina) to use electron microscopy to characterize the protein-DNA complexes formed in vitro during DNA replication by the T4 proteins. - DNA Replication, DNA polymerase, Helicase, Primase, gene 59 helicase assembly protein, RNase H, Bacteriophage T4, crystal structures